Enzymatic omega carboxylation of fatty acids



United States Patent @fillCd Bflhdibh Patented Mar. '5, lQfiS 3,080,296 ENZYMATEC OMEGA. (IAREGXYLATEGN UF FATTY AGES Kenneth C. Robbins, Chicago, lib, assignor, by rnesne assignrnents, to Armour Pharmaceutical ilornpany, a corporation of Delaware No Drawing. Filed May 5, 1969, Ser. No. 26,955 12 Claims. tfl. 195-40) This invention relates to the enzymatic oxidation of monocarboxylic acids. More particularly this invention relates to the direct in vitro preparation of straight chain terminal dicarboxylic acids from corresponding unbranched long chain monocarboxylic acids catalyzed by animal visceral organ homogenates.

The direct, one step, conversion of monocarboxylic acids having 8 to 11 carbon atoms to the corresponding dicarboxylic acids by omega oxidation occurs in man and dog, in vivo. Omega oxidation refers to the methyl oxidation of the paraffin end of the fatty acid molecule. The following illustrates this reaction:

X represents an integer of 3 to 6.

Omega oxidation in vivo competes with beta oxidation, beta oxidation referring to oxidation of the carbon atom second from the carboxyl group of a monocarboxylic acid. In vivo omega oxidation apparently occurs only under emergency conditions, since formation of the dicarboxylic acids occurs over a very narrow range of fatty acids. Beta oxidation is thought to be the usual in vivo mode of fatty acid oxidation.

dethyl end groups of main chains of branched fatty acids can be readily oxidized to dicarboxylic acids provided the main chains are of medium length. Branched unsaturated fatty acids, if in the form of amides, can also be made to undergo similar terminal oxidation. How ever, heretofore the direct or single step oxidation of the terminal methyl group or" a straight chain fatty acid has not been accomplished by in-vitro organic chemistry techniques. Such a direct, one-step, oxidation is of great practical interest. It could be employed in methods for preparing new fatty acid derivatives of fatty acids difficult to synthesize. In addition known compounds may be prepared more economically.

It is therefore a general object of this invention to provide a method of enzymatically oxidizing terminal methyl groups of straight chain monocarboxylic acids.

A more specific object is to provide a method of enzymatically convertin" terminally saturated unbranched long chain monocarboxylic acids to their corresponding terminal dicarboxylic acids.

Briefly the process or" this invention comprises contacting a normal terminally saturated straight chain monocarboxylic acid with animal tissue homogenates. Contacting such monocarboxylic acids with animal tissue homogenates results in carboxylation of the terminal methyl groups.

The broken cell homogenates essential to this process are derived from animal tissue. In accordance with my findings, most animal tissues probably contain enzyme displaying some omega oxidation activity. However practical sources of these enzymes systems are tissues having a high rate of metabolic activity. These include organ and gland tissues such as heart, spleen, liver, kidney, stomach, gonads and so on. The visceral or splanchnic organs and glands are the richest sources of these enzyme systems.

The enzyme system contained in such tissues and which catalyzes the oxidation of the terminal carbon of the fatty acid may be described as an omega fatty acid oxidase. Measurable quantities of monocarboxylic acid are converted to the dicarboxylic acid simply by contact with whole tissue homogenates. However, beta oxidation is competing with the omega oxidation system whole tissue is used. Beta fatty acid oxidase appears to be localized in the cell mitochondria. Coenzyme A is a necessary cofactor for potentiation of the beta -oxidation enzyme system. Even though the mitochondria is retained in the homogenate there is such a low level of naturally occurring coenzyme A that omega oxidation will proceed at a measurable rate.

Animal visceral organ tissue homogenates are the richcst sources of enzymatic activity necessary for catalysis of omega oxidation. A homo-genate is a broken cell suspension. Any of the usual methods of disrupting cells such as mechanical grinding or treatment with certain organic solvents may be employed. Preferably the tissue homogenate is suspended in a no-nelectrolytic solution. Electrolytes cause agglutination of microsornes and mitochondria. lsotonic sucrose serves as an excellent medium for the broken cell suspension. The homogenates are preferably fractionated to obtain maximum catalytic activity. Removal of the mitochondrial fraction increases the rate of omega oxidation by removing enzyme systems catalyzing competing beta oxidation. Any of the classical fractionation methods may be employed. Differential centrifngation is the preferred fractionation method.

The niicrosonie and soluble fractions of the cell homogenates contain the bulk of the omega fatty acid oxidase system required by the process of this invention.

One method of preparing a homogenate yielding a preparation having excellent conversion activity is the following. Visceral organs, hog livers or dog kidneys for example, are chilled in 0.25 M sucrose at 0 C. The chilled organs after draining are minced. One part of tissue is suspended in about 9 parts of cold 0.25 M sucrose solution. Preferably a temperature of 0 to 4 C. is maintained throughout all fractionation steps. This tissue preparation is then homogenized for 2 minutes in a Potter-Elvehjem homogenizer in which a fitted cylindrical nestle is rotated within a tube.

The homogenized preparation is then subjected to dif- :ferential centrifugation. Con-trifugation at between 10,000 to 20,000 g, g being the force of gravity, for 10 minutes results in removal of cell nuclear and mitochondria fractions. The supernatant which contains microsornal and soluble fractions is essential to omega oxidation. At centrifugal forces approaching 54,000Xg the microsomal cell fraction is removed from the supernatant. Removal of this microsomal fraction drastically reduces activity of the preparation. Therefore it is necessary that centrifugal force of at least 10,000X g but less han 50,000 ,5; should be employed. Although not essential, dialysis of the microsome and soluble fractions against 4-tris hydroxyrnethyl amino methane butler, known as Tris buffer, gi es a preparation having high conversion activity.

in practicing this invention large scale methods of preparing tissue homogenates and subjecting these homogenates to differential centrifugal separation are ordinarily required. One such method of preparing large quantities of tissue particulates is the following procedure. Fresh porcine or bovine livers, examples of tissue containing rich sources of desired enzyme systems, are Washed in ice cold 0.15 M NaCl and cut into small pieces. Grinding in a refrigerated standard meat chopper through a /8 inch plate serves to disrupt tissue cells. Regrinding gives better results. A 10% suspension in appropriate suspending media is desirable. A satisfactory suspendosa es ing medium is 0.25 M sucrose containing 0.001 M versene (ethylene diarnine-tetra-acetic acid. The suspension is stirred at about 2 C. for from 15 to 30 minutes. After filtering or settling for 15 minutes the suspension is decanted to remove most of the unbroken cells and cell debris before centrifugation.

The liver homogenate after settling, or filtration, is centrifuged in an International centrifuge, size 3, No. 258 head at 1250 g (2,000 rpm.) for 15 minutes at 4 C. The resulting supernatant is then further centrifuged in a Model T-l Sharples super centrifuge at 13,200 g (23,000 rpm.) using a standard clarifier rotor ('11 H) at a feed rate of up to 100 ml. per minute at 4 to 6 C. The precipitate contains the mitochondrial fraction. This large scale differential centrifugation method can be operated continuously to prepare cell particulates and soluble fractions.

After removal of the mitochondrial fraction the supernatant contains the soluble and microsomal fractions. For characterization purposes the microsomal fraction may be separated from the soluble fraction by clumping methods. To separate the microsomes, CaCl solution is added to the supernatant fraction to give a final concentration of 0.01 M CaCl The CaCl; clumps the residual particulates suspended in the supernatant allowing them to be more easily removed by centrifugation. After allowing the CaCl treated supernatant to stand for l to 2 hours the suspension is centrifuged in the Sharples centrifuge at 13,200 g in a clarifier rotor at 4 to 6 C. The precipitate removed by this procedure may be called the mic-rosomal fraction. The resulting supernatant may be called the soluble fraction. The microsome fraction resulting from this separation technique contains 35% to 41% protein. The soluble fraction contains about 5% to 6% protein. The bulk of the non-protein material in both fractions is lipid in nature.

Terminally saturated straight chain monocarboxylic acids having a carboXyl group attached to the alpha carbon atom, normal position, are the compounds converted by the process of this invention. Salts and esters of these acids are also suitable substrates. Monocarboxylic acids having 8 to 18 carbon atoms may be converted by this process. I prefer to use fatty acids having 9 to 13 carbon atoms however. Unsaturated as well as saturated straight chain fatty acids are suitable starting materials for this conversion. It is however essential that the terminal carbon atom be saturated.

Fatty acids resulting from the hydrolysis of natural fats and oils may also be converted to dicarboxylic acids by this process.

Examples of suitable starting materials for this process are (1) saturated acids such as caprylic, pelargonic, capric, hendecanoic, lauric, tridecan'oic, myristic, pentadecanoic, palmitic acids; (2) unbranched unsaturated acids which are terminally saturated such as A decylenic acid, oleic, palmitoleic, and linoleic acids; (3) natural products such as fatty acids obtained from meat fats and palm, coconut, cottonseed, lard and fish oils.

The in vitro conversion process comprises contacting a suitable monocarboxylic acid with an animal tissue homogenate. Preferably the tissue homogenate is reduced mitochondria-free. And preferably certain cofactors are added to potentiate conversion activity of the omega fatty acid oxidase enzyme system contained within the animal organ tissue homogenate.

In general the conversion in its preferred form comprises mixing a fatty acid of the hereinbefore described type with the microsomal and soluble fractions of a visceral organ tissue homogenate. Fatty acid concentration is preferably in the range of 0.03 to 6 micromoles per milliliter. The operable range is however far beyond these limits.

Concentrations of the required subcellular fractions, including the microsome and soluble fractions, may vary widel". Whole tissue homogenate which is undiluted may be directly mixed with the fatty acid starting material. However, a Whole tissue homogenate concentration of 2 to 4 percent is preferred. Higher concentrations will to some extent increase rate of conversion. Equivalent amounts of the critical subcellular fractions extracted from tissue representing from 2 to 4% of the reaction system will be kinetically equivalent.

A pH range of 6.5 to 9.5 is considered optimum. However, a range of 5.3 to 10 is operable. Attempts to react outside this pH range make denaturization of the tissue particulates imminent.

Conversion may be accomplished at temperatures above 0 C. and below 60 C. A temperature of 37 C. is however preferred. Temperatures above 60 C. will result in gradual inactivation of the enzyme systems contained in the subcellular particulates. Temperatures below 37 C. impede rate of reaction.

This conversion may be described as an aerobic reaction. It is therefore apparent that aerobic conditions should be established for best yields.

Using optimum levels of all the above described conditions reaction time will be approximately 30 to 60 minutes.

Certain cofactors are preferably added to the homogenates for maximum enzymatic conversion activity. These cofactors include magnesium ions (Mg++), diphosphopyridine nucleotide (DPN) and triphosphopyridine nucleotide (TPN). The reduced form of TPN (TPNH) is somewhat more effective than the oxidized form of TPN (TPN- I prefer to use a Mg++ concentration of 0.015% based on the whole tissue homogenate Weight. I also prefer to use a nucleotide concentration of 0.0024% of both DPN+ and TPNH based on the weight of the whole tissue homogenate. Concentrations greatly in excess of these levels of Mg++, DPN+, and TPNH are obviously not detrimental to the conversion. Use of cofactors is not essential to the conversion. Measurable quantities of dicarboxylic acid may be prepared by contacting monocarboxylic acids with undiluted and unbuffered tissue homogenates containing soluble and microsomal fractions.

The following specific examples will serve to further describe the above generally outlined principles of this invention.

Example I A 10% hog liver tissue homogenate was prepared. The homogenate consisted of 1 part tissue in 9 parts of isotonic sucrose. Precursor monocarboxylic fatty acids were labelled with radioactive carbon C so that conversion to the corresponding dicarboxylic acids could be verified by use of isotope tracer techniques. Three ml. systems consisting of the following were prepared: 70 ,uM, pH 7.4, 0.1 M phosphate buffer; 1 M monocarboxylic acid (C 1.0 ml. 10% tissue homogenate; 6 ,uM adenosine triphosphate (ATP); 15 M Mg' 2.4 ,uM D'PN 2.4 M TPN 60 ,uM nicotinamide. The reaction was carried out at 37 C. for 60 minutes using a Burrell shaker.

Isotopic tracer techniques and gas chromatography assay methods were used to measure the end-product straight chain dicarboxylic acids.

0.01 ,uM dicarboxylic acid is equivalent to 1,000 conversion units in the following table:

Example II Various visceral organ tissue homogenates were prepared. These were contacted with labelled radioactive C caprylic acid. The conditions and cofactors were identical to those described in Example 1:

Units Converted Per Hour Species 'Tissue Example III Using the same system described in Example I, invitro conversion of decanoic acid to .decanedioic acid by hog liver subcellular fractions was accomplished. The cell particulates were prepared by differential centrifugation.

The mitochondria fraction Was isolated by the following method: Centrifugation for minutes at 700 g, resulting supernatant centrifuged for 10 minutes at 5,000 g, resulting pellet redispersed by homogenization in 0.25 M sucrose, redispersed pellet centrifuged at 24,000 g for 10 minutes. The microsome fraction was isolated by the following technique: Combined supernatants obtained in isolation of the mitochondria were made up to original volume with 0.25 M sucrose centrifuged for 60 minutes at 54,000 g. The resulting pellet contains the microsomal fraction. The supernatant contains the soluble fraction.

1 hit-Mitochondria; McMicrosomes; S-Soluble fraction.

Example IV Certain cofactors have a marked effect on efiiciency of conversion of monocarboxylic acids to corresponding dicarboxylic acids.

Decanoic acid was oxidized to sebacic acid by dialyzed hog liver tissue microsomes and subcellular soluble fractions. The subcellular fractions were prepared by differential ccntrifugation and dialyzed against 0.05 M pH 7.4 tris-hydroxymethyl arninomethane bufi'er (Tris-buffer), 5 ,uM nicotinamide isocitrate, and 2 ;LM TPNH.

The 3 milliliter systems with all oofactors present consisted of the following: 200 ,uM, pH 7.4, 0.2 M Tris bufier; 1 ,uM radioactive monocarboxylic acid (C 2 M dicarboxylic acid; microsomal and soluble fractions equivalent to 1 gm. tissue; 15 nM Mg++; 60 ,ILM ATP; 2.4 ,u.l\/I DPN' 2.4 M TPN 60 ,uM nicotinamide. Reacted for one hour at 37 C. on a Burrell shaker. In experiment (2) soluble fraction was dialyzed against water.

Labelled radioactive, C decanoic acid was the precursor monocarboxylic acid. Isotopic tracer techniques of assaying the radioactive end-product, suberic acid, in the carrier pool of dicarboxylic acid were employed. 0.01 M of labelled dicarboxylic acid is equivalent to 1,000 units in the following table.

From these data it becomes apparent that Mg DPN+ and TPNH or TPN+ are important cofactors for providing efficient conversion.

The foregoing detailed description and examples are given only for clearness of understanding, and I do not desire to be limited to the exact details given, for obvious modifications may be made by one skilled in the art Without departing from .the spirit of this invention.

I claim:

1. The process of in vitro oxidation of monocarboxylic acids to corresponding straight chain non-substituted terminal dicarboxylic acids by contacting terminally saturated straight chain normal monocarboxylic acids having a ca-rboxyl group attached to the carbon atom, normal position, and having 8 to 18 carbon atoms with animal visceral organ tissue homogenates having omega oxidation activity and being substantially mitochondria-free.

2. The process of in vitro enzymatic omega oxidation of terminally saturated long chain non-substituted monocarboxylic acids to the corresponding dicarboxylic acids by contacting monocarboxylic acids having a oarboxyl group attached to the carbon atom, normal position, and having 8 to 18 carbon atoms with animal tissue cell microsomal and soluble fractions.

3. The process of converting terminally saturated straight chain non-substituted monocarboxylic acids having a carboxyl group attached to the carbon atom, normal position, and having 8 to 18 carbon atoms to the cor-responding dicarboxylic acids by contacting said monocarboxylic acids with animal visceral organ tissue fractions having omega oxidation activity and remaining in the supernatant of a broken cell suspension subjected to centrifugal forces of at least 10,000 but not exceeding 50,000 times the force of gravity.

4. The process of in vitro oxidation of saturated straight chain non-substituted normal monocarboxylic acids having a carboxyl group attached to the carbon atom, normal position, and having 8 to 18 carbon atoms to form corresponding straight chain terminal dicarboxylic acids comprising the steps of contacting said monocarboxylic acids with animal visceral organs cell fractions having omega oxidation activity and remaining in a broken cell suspension subjected to centrifugation at a force of 10,000 g to thereby catalyze said oxidation, and mixing cofactors with said cell fractions, said cofactors being selected from a group consisting of magnesium ions, triphosphopyridine nucleotide and diphosphopyridine nucleotide.

5. The process of converting terminally saturated straight chain non substituted normal monocarboxylic acids having a carboxyl group attached to the carbon atom, normal position, and having 9 to 13 carbon atoms to the corresponding straight chain terminal dicarboxylic acids by contacting said monocarboxylic acids with animal visceral organ homogen-ate fractions consisting essentially of soluble fractions and microsomes, said fractions being activated by cofactor-s, said cofactors being selected from a class consisting of magnesium ions, tripho-sphopyridine nucleotide and diphosphopyridine nu cleotide.

6. The process of claim 5 wherein caprylic acid i s converted to suberic acid.

7. The process of claim 5 wherein pelargonic acid is converted to azelaic acid.

8. The process of claim 5 wherein capric acid is converted to seoacic acid. r v

9. The process of claim 5 wherein hendecanoic acid is converted to hendecanedioic acid.

10. The process of claim 5 wherein lauric acid is the starting material.

11. The process of converting non-terminally unsaturated straight chain non-substituted monocarboxylic acids having a carboxyl group attached'to the carbon atom, normal position, and having 8 to 18 carbon atoms to the corresponding straight chain terminal 'dicarboxylic acids by contacting said monoca-rboxylic acids with animal visceral organ homogenate subcellular fractions consisting essentially of the soluble and mi-crosomal fractions, and cof'actors, said cofactors being selected from a class consisting of magnesium ions, triphosphopyridine nucleotide, and diphosphopyridine nucleotide.

12. The process of converting terminally saturated straight chain non-substituted monocarboxylic acids having a carboxyl group attached to the carbon atom, normal position, and having 8 to 18 carbon atoms to the corresponding straight chain terminal dicarboxylic acids by contacting said monoc-arboxylic acids with animal Visceral organ tissue homogenates having omega oxidation activity and which have been rendered substantially mitochondria-free.

References Cited in the file of this patent Karrer: Organic Chemistry, 4th ed., Elsevier Publ. Co., Inc, N.Y. (1950), page 198.

Sumner et al.: Chemistry and Methods of Enzymes, 3rd ed., Academic Press Inc. Publishers, N.Y. (1953), page 208.

Downes: The Chemistry of Living Cells, Harper and Brothers Publishers, N.Y. (1955), pages 491, 494 and 495.

Deuel: Lipids, vol. III, Interscience Pub. Inc., N.Y. (1957), pages 87 to 90. 

1. THE PROCESS OF IN VITRO OXIDATION OF MONOCARBOXYLIC ACIDS TO CORRESPONDING STRAIGHT CHAIN NON-SUBSTITUTED TERMINAL DICARBOXYLIC ACIDS BY CONTACTING TERMINALLY SATURATED STRAIGHT CHAIN NORMAL MONOCARBOXYLIC ACIDS HAVING A CARBOXYL GROUP ATTACHED TO THE CARBON ATOM, NORMAL POSITION, AND HAVING 8 TO 18 CARBON ATOMS WITH ANIMAL VISCERAL ORGAN TISSUE HOMOGENATES HAVING OMEGA OXIDATION ACTIVITY AND BEING SUBSTANTIALLY MITOCHONDRIA-FREE. 